Cell fractionator apparatus and method



2 Sheets-Sheet 1 Jan. 12, 1965 J. BLUM ETAL CELL FRACTIONATOR APPARATUSAND METHOD Filed. Feb. 7, 1962 g (Q wt $774,945: J 742 .6 ATTOR ti-EY i.Q\\ m mm mm 6.9 I \RR M m \Q Q an Nu m wn m Q LUL Q Q P Q Q 1N H Jan.12, 1965 J. BLUM ETAL CELL FRACTIONATOR APPARATUS AND METHOD Filed Feb.7, 1962 2 Sheets-Sheet 2 INVEN'QRS (iv/was J F42 BY 2 z 5 ATTORNEYUnited States Patent 3,165,266 1 CELL FRACTIONATOR APPARATUS AND METHODJosef Blum, Norwalk, and Charles J. Filz, Weston, Conn,

assignors to Ivan Sorvall, Inc, Norwalk, Conn, a corporation ofConnecticut Filed Feb. 7, 1962, Ser. No. 171,674 19 Claims. (Cl. 241-4This invention relates to an improved apparatus and method for rupturingcells in order to study the structure and function of their constituentparts.

For a long time it has been realized that research in the areaencompassing structure and function of microorganisms would be greatlyfacilitated if it were possible to rupture the cells and isolate thestructural elements such as cell walls and protoplasm withoutsignificant alteration of these components. During the past ten years agreat deal of attention has been directed toward development of toolsand techniques for this purpose, and several instruments have beenproduced. Shaking and grinding devices, including the ultrasonicoscillator, have been used to disrupt'microorganisms but when separationof the disruption products was attempted, a portion of the 'cell wallswere fragmented to such an extent that it was impossible completely toremove them from the protoplasrn of differential centrifugation. Thus,the yields of pure cell wall material were small in proportion to theamount of starting material, and protoplasm substantially free of cellwall material could not be produced. Neither cell walls nor protoplasmcould be isolated in appreciable amounts from Mycobacteria (tuberclebacilli) by the use of these devices due to the tendency of the brokenand unbroken cells and the protoplasm to aggregate to a butterlikeconsistency.

The French pressure cell, in which a pressurized microbial suspensionisreleased at an orifice, seemed to be the most logical approach to theproblem, since pressures could be selected and regulated according tothe rigidity of the cell walls of a given microorganism. The cells wouldcrack as they emerged through the orifice and would not be subjected tofurther disintegration. When this method was used, recovery of cell wallmaterial was quite satisfactory, but temperatures at the orificeincreased to a point whereQpartial denaturation of certain protoplasmicconstituents occurred, and this material adhered to the inner surface ofthe cell wall and could not be'r'emoved. Conventional cooling methodswere not effective.

It was then proposed that the orifice and needle valve of the pressurecell be exposed to a stream of pre-cooled gas that was released from aseparate needle valve. While this made it possible to produce somewhatbetter results in the form of purified cell walls and protoplasm, it Wasnot until the production of the apparatus and method of the presentinvention that suitably controlled results in quantity could beobtained.

The present invention comprises an improved needle valve assembly andexpansion valve seat in conjunction with an improved cooling system bywhich the fractionated components of cells in the form of readilyseparable cell walls and protoplasm may be recovered in proper conditionand in sufficiently large quantities for study and research.

Amongst the features of the improved apparatus and ice to preserve thecharacteristics of the fractionated components of the cells; (b) aspecially designed needle jvalve structure for controlled bleeding ofthe pressurized suspension; (c) an improved valve assembly structure fortransmitting the compressed suspension into the valve chamber; and (d)means for utilizing asingle refrigerating system for cooling thecompressor of the cell suspension and utilizing the same cooling mediumwhich is in the form of an inert gas to cool the expansion chamher andthe needle valve orifice. b

Still other objects and advantages of the invention will be apparentfrom the specification. I I

The features of novelty which are believed to be characteristic of theinvention are set forth herein and will best be understood, both as totheir fundamental principles and as to the particular embodiments, byreference to the specification and accompanying drawing, in which:

FIGURE 1 is a vertical central section view of the improvedfraction'ating valve system and the outlet orifice cooling arrangementtherefor, some parts being shown in partial elevation, and some partsbeing broken away;

FIG. 2 is a greatly enlarged section view of a portion of the apparatusshown in FIG. 1;

FIG. 3 is a further enlarged view of a portion of the apparatus, takenon line 3-3 of FIG. 2; and

FIG. 4 is a schematic representation, in reduced size, but not inrelative scale, of the complete cell fractionating system of which thevalve apparatus shown in FIG. '1 forms an integral part.

Referring to the drawings, the cell fractionating process is shownschematically in FIG. 4, where a specimen l1 constituting such materialsas bacteria, fungi, spores, viruses, blood cells, and the like, inliquid suspension, is contained in a supply bottle 12. The specimen iscultured in the normal manner in nutrients which act as the liquidvehicle. Inserted into supply bottle 12 is one end of a flexible tube13, made of rubber, neoprene, or the like, the other end of which isconnected to the inlet port of high pressure one-way valve 14 which isadjusted by means of control rod 16, the outer end of which is providedwith a manually or mechanically operable knob 17.

Connected to theoutlet port of valve 14 is one end of transfer tube 18,the other end of which is connected to the inlet port of pressure cell19. Pressure cell 19 has a cylinder 21 which slidably accommodates aplunger 22 connected to ram 23 mounted on piston 24 slidably positionedwithin hydraulic cylinder 26. Upon the outward motion of plunger 22 frompressure cell 19, suction is created whereby a specimen is drawn from aspecimen supply bottle 12 through valve 14 and into the cavity ofcylinder 21 of said cell. of plunger 22 into cylinder 21 pressure isapplied to the specimen in the cavity of the pressure cell, causing thespecimen to pass through transfer tube 28, one end of which communicateswith cylinder 21, the other end of which communicates with the interiorof the expansion method herein are: (a) an improvedvalve assemblystrucating expansion chamber with means for continuously cooling saidexpansion chamber and said orifice in order valve assembly, generallydesignated 29, thedetailed structure of which is shown in FIGS. 1, 2 and3.. Upon being processed through the expansion valve 29, thefractionated cells then pass through tube 31 into a receiver bottle 32into which a or 200 cc. of a suspending medium has been placed.Thereafter, the materials in the receiver bottle may be transferred to acentrifuge where the components are physically separated for suchfurther study as may be required.

Tube 31 extends through a rubber stopper 33, or the like, in the mouthof the receiver bottle 32, said stopper also having a venting tube 34extending therethrough to facilitate the filling of the bottle.

- The control system for the hydraulic cylinder 26 comprises a sump orreservoir 36 of hydraulic liquid. A suction pipe line 37, extending intosump 36, is connected Upon the inward movement to a suitable pump 38which causes the hydraulic fluid to pass by way of pipe line 39 througha three position valve 41, which is operated by a pair of solenoids 42and 43. The solenoids are actuated by suitable electric circuitry whichis well known in the art, and which does 'not constitute any part of thepresent invention. Valve 41 is shown with the right hand section Apermitting free flow of hydraulic fluid to and from hydraulic cylinder26. The central section B ofvalve 41 is the no-go position, while theleft hand section C has a cross-over arrangement for reversing-the flowofthe hydraulic'fluid.

In the position of the valve 41 shown in FIG. 4, hydraulic fluid thenpasses under pressure by way of pipe line 44 through filter 46 and byway of pipe line 47 into a flow control valve 48 which determines therate fiow of the hydraulic fluid through pipe line 49 into hydrauliccylinder 26.

It will be noted that pipe line 39 is provided with relief pipe line 51communicating with relief valve 52, the pressure control actionof whichis represented schematically by spring 53, whereby excessive pressurecan be relieved by permitting flow of hydraulic liquid through outletpipe 54 into the sump 36. Flow control valve 48 is also provided with anoverflow pipe line 56 which also leads back to the sump 36.

The introduction of hydraulic fluid under pressure through pipeline 49into cylinder 26 is operative by way of piston 24 and ram 23 to driveplunger 22 into pressure cell cylinder 21. This takes place when valve41 is in the position shown in FIG. 4. Overflow of hydraulic fluid fromcylinder 26 during the pressure stroke returns through pipe lines 57 and58 to sump 36. A retracting or suction stroke of plunger 22 is producedby moving valve 41 to the right into the C position, whereby the flow ofhydraulic fluid is reversed and pipe line 57 becomes the pressure line,pipe line 49 becomes the return line, and pipe line 56 is the overflowline into sump 36. In the B position of valve 41,.the action of thehydraulic cylinder 26 is at a standstill, and if pump 38 continues tooperate, the hydraulic fluid simply returns to sump 36 by way of pipeline 58.

On the suction stroke of plunger 22, a specimenis drawn from supplybottle into cylinder 21 of the pressure cell. is transmitted throughpipe 28 to the expansion valve 29, the return of the specimen to'supplybottle 12 being prevented by one-way valve 14.

A double purpose cooling system isprovided in order to maintain pressurecell 21 and expansion valve 29 at the requisite low temperatures, sinceconsiderable heat is generated by the operation of both of these piecesof apparatus.- The cooling medium'consisting of dry nitrogen, air,carbon dioxide, or other neutral medium, is storedin supply tank 61which is provided with suitable pressure maintaining apparatus, notshown. The refrigerating cir- On the pressure stroke of plunger 22, thespecimen cuit, shown only in part in FIG. 4, consists of a cooling 7then passes through pipe 73 into the expansion valve assembly 29 whereit maintains the requisite low'temperature at-theexit orifice oftheruptured cells. Thence the gas and the cells pass through tube 31into the receiver bottle 32, said gas then escaping into the atmospherethrough vent tube 34.

As shown in FIGS. l3, the expansion valve assembly comprises a body 76,to the top of which is connected a nut 77 bygmeans of a, plurality ofbolts 78, and to the bottom of which is connected a bottom plate 79;, bymeans of a plurality of bolts 81. Body 76, nut 77, and bottom plate 79are made of steelor the like, and may be circular in cross-section. Inone embodiment their diameter is in the order of about 2 /2".

Nut 77 has a vertical central threaded aperture 82 which accommodates athreaded valve stem screw 83, the upper portion of which extends abovethe top of said nut. Valve stem screw 83 has a .central, longitudinalbore 84, the upper end of which terminates in a threaded coaxialaperture 86 of somewhat greater diameter, which accommodates set screw87.

Bore 84 in valve stem screw 83 movably accommodates an elongated valvestem 88, which also extends into bore 89 in body 76, bore 89 beingcoaxially aligned with bore 84. The lower end of valve stem 88terminates in a conical valve head 91, whose function willbe describedhereinafter. The upper end of valve stem 88 abuts the lower end of setscrew. 87 which can be adjusted to compensate for wear on valve head 91,and to maintain valve stem 88in proper position in respect of stem screw83. The relative dimensions of valve stem 88 and of bore 84 of stemscrew 83 provide a barely running fit with approximately one,10-thousandths inch (.0001") clearance therebetween.

The lower end of bore 89 in body 76 terminates in a coaxial circularbore 92 of somewhat greater; diameter. The lower end of bore 92communicates with a stilllarger coaxial, circular recess 93 whichaccommodates expansion valve seat block 94 which is maintained captivetherein by bottom plate 79. f

Block 94 has an integral upwardly extending central tubular boss 96 thatfits closely into bore 912. Bore 92 has an annular recess 97 whichaccommodates resilient O-ring 98 made of a suitable material such, asrubber, neoprene, nylon or the like, which serves as a seal vbetweenboss 96 and body 76. Positioned at the top of boss 96 is an 0- ring 99,also made of similar resilient material, which serves to sealvalve stem88 whichadjacent parts. 'The sealing action of O-rings 98 and 99 isenhanced by the pressure conditions obtaining .in the system when theapparatus is in operation as will be described hereinafter.

The lower end of valve stem 88 extendscoaxially into the interior ofvalve chamber 101 in tubular boss 96 and block 94, there being a slightclearance between the surface of said stem and the interior wall of saidchamber suflicient to permit liquid specimens to'pass freely betweensaid elements to fill valve chamber 101.. Intermediate its ends, boss 96has a plurality of radially and equidistantly arrayed ports 102 (FIGS-:2and 3), the outer countersunk ends of which communicate with an annularrecess 103 in body 76. Recess 103 serves to distribute specimenmaterials. substantially. equally through ports 102 into valve chamber101. Distributor recess 103 communicates with a horizontal specimentransmitting channel 104 which extends to threaded inlet recess 106 invbody76.

7 Since the specimen is delivered from'cell'19 (FIG. 4) to the expansionvalve assembly 29 under high'pressure, pipe line 28 is made of ,asubstantially rigid material, which, in one embodiment, comprises a 6inch diameter pipe having a 16th inch inside diameter. Pipe 28 has ;aground tapered end 167 which mates with a ground tapered seal 108attheinner end of recess 106 in body 76.

In order to ensure an intimate leak-proof fit between pipe end 107 andseat 108, theendportion of pipe'28 is threaded to accommodate a threadedferrule 109. The

annular wall of inlet recess 106'is threaded to accommodate threadedgland 111 which has a circular aperture 112 into which ferrule 109 fits.Gland 111 has an outwardly extending integral rim 113 to facilitaterotation thereof whereby the interior wall 114 of aperture 112 is forcedagainst the end offerrule .109 to cause tapered end 107 of pipe 28 to beurged into firm and intimate contact with seat 108. By this means a highpressure leak-proof connection is established between the interior ofpipe 28 and channel 104 in body 76.

.Similar high pressure, leak-proof connections are provided betweenpressure cell 19 and pipes 18 and 28, respectively and between pipe 18and high pressure unidirectional valve 14. These high pressureconnections have a sufficient safety margin in strength to withstand thehigh pressure forces that are generatedwithin pressure cell 19.

The lower end of transfer chamber 101 in block'94 has a conical orconcave conoidal constriction 116 which terminates in a small circularneck'117 which, in turn, communicates coaxially with, and forms thesmaller end of, a conical open-mouthed expansion chamber 118. When valvestem screw 83 is turned to cause the lower portion of conical valve head91 to extend through neck 117, whereby an annular portion of valve head91 bears firmly against an annular portion of constriction 116 whichserves as a valve seat for said valve head, a leak-tight seal is formedwhich withstands the high pressures generated by pressure cell 19,thereby preventing the escape of the specimen from transfer chamber 101into expansion chamber 118. a

The mouth of expansion chamber 118 opens out upon a coaxial bore 119 inbottom plate 79. Communicating with bore 119 is a horizontal channel 121which terminates in a coaxial widened recess 122 which accommodates theinner portion of an inlet nipple 123. Connected to nipple 123 is pipeline 73 (FIG. 4) by which cooled nitrogen or other neutral gas istransmitted to bore 119. The lower portion of bore 119 is fitted with anoutlet nipple 124, to the lower end of which is connected tube 31leading into the specimen collection bottle 32.

Nipple 124 has an intgeral inner tubular end portion 126 which extendsfreely into bore 119 whose diameter is somewhat larger than tube 126,the upper end of said tube being spaced apart from the bottom of block94 to permitcommun-ication between bore 119 and expansion chamber 118.

The cold nitrogen passing through channel 121 flows I continuously intobore 119 around tube 126, and circulates upwardly into expansion chamber118 to cool said chamber, the lower portion of valve head 91, and thatportion of constriction 116 that forms a valve seat for valve head 91. a

In operation, the specimen materials in supply bottle 12 are drawn intocylinder 21 of pressure cell 19 by the retraction of plunger 22, withvalve 41 in the position where the C-portion thereof is arrayed betweenpipe lines 39-44 and 57-58. Thereafter, valve 41 is moved into the Aposition, as shown in FIG. 4, whereby the hydraulic system is operativeto cause plunger 22 to produce its pressure stroke in cylinder 21,thereby bringing the materials in said cylinder under high compression.One-way valve 14 prevents the specimen materials from returning intosupply bottle 12 while the forward end of the system including valvechamber 101 is closed by valve head 91- bearing firmly against valveseat 116.

When the requisite pressure has been achieved upon the specimen in theclosed system, this is indicated by a suitable pressure gauge 131, orthe like, mounted in pipe line 49. Gauge 131 is calibrated to read thepressures in the closed specimen system by simple computation thatestablishes the relationship between the pressures imposed upon thelarge piston 24 in hydraulic cylinder 26;, and the pressure translatedthereby through smaller plunger 22 in pressure cell 19.

Thereafter valve stem 88 is retracted very slightly by rotation of screw88 to barely back ofl? valve head 91 from constriction 116 by a distancein the order of a few thousandths of an inch, to form an annular orificebetween valve head 91 and constriction 116 through which the specimenescapes from chamber 101. Upon passing through said orifice and throughneck 117, the specimen is suddenly subjected tolexplosive decompressionin expansion chamber 118. Assisted by the flow of the cooling 6 gas, theexploded cells then move by gravity into and through tube 126 and outletneedle 124, and thence through tube 31 into receiving bottle 32.

Since tremendous energy is released in pressure cell 19 due to thecompression of themateri als in cylinder 21, which is one embodiment,reaches as much as 57,000 pounds per square inch, the cooling. mediumfrom the refrigerating system is circulated around cell 19 through coil69'to absorb the heat generated and to chill the cell, and to keepcylinder 21 cool.

It is vitally important to keep the specimen cool as it undergoesdecompressiomtherefore, the cooling medium is conducted back into therefrigerating system through pipe 71 into heat exchange 63 forre-ccoling, and thence valve area in block 94 is maintained at atemperature whereby the specimen emerging from said decompressionchamber at no time exceeds a temperature of approximately 10 C., in oneembodiment.

By providing for a continuously circulating stream of cold gas withinthe critical valve area, and within'the decompression chamber, said gasmixing intimately with the fractionated cells as they emerge from thevalve chamber 101, it is possible by means of the apparatus and systemdisclosed herein, to preserve the fractionated cell particles andconstituents in a proper condition for further study,examinatiomanalysis and experimentation.

The intimately mixed fractionated cells and cooling medium passdownwardly through outlet nipple 124 and through pipe 31 into receivingbottle 32, where the specimens in liquid suspension are collected. Thegases of the cooling medium passing into said bottle escape therefromthrough vent tube 34.

By providing for the conical valve head 91 to protrude throughconstriction 116 and through neck 117 where the materials under highpressure escape through an annular orifice, directly into a coaxialexpansion chamber, not only is the controlled retraction of valve head91 facilita-ted, but also it is now possible to ensure the controlledcooling of the critical area in which the specimens under high pressureundergo explosive decompression or expansion. This was hithertoincapable of being achieved where the specimen under pressure flowed ina direction opposite to the valve head, thereby preventing access of acooling medium to the valve head and the valve seat, in order topreserve the integrity of the specimen structures. Also, by providingfor specimen flow in the same direction as the valve head, into acoaxial expansion chamber, the specimen does not impinge upon anyobstruction to its flow which would otherwise denaturize thefractionated material.

The arrangement of making the valve stem structure in two parts, namely,stem 88 and screw 83, constitutes an improvement over a one-partstructure. Due to the precision requirements of providing the controlledminute orifice between valve head 91 and constriction 116 with accurateconcentricity of the conical end of said valve head in respect to valveseat and of neck 117 for even distribution of the specimen, it has beenfound that valve stem 88 and its head 91 can more readily be accuratelymachined as a part separatefrom valve stem screw 83, whose bore 84, canalso be more accurately machined in relation to bore 89 in body 76. Therelative positions of valve stem 88 and valve screw 83 to each other areestablished by set screw 87.

After the requisite pressure of the specimen has been achieved in thesystem, valve head 91 is very slightly retracted from constriction 116by rotating valve stem screw 83 a small fraction of a turn. While thespecimen is escaping through neck 117, the pressures within the systemare sulficient to cause the specimen in valve chamshown.

ber 101 to bear upon valve head 91 and to urge valve stem 88 upwardlyagainst the lowerend of set screw 87, whereby the requisite position ofsaid valve stem 88 relative to the position of valve stem screw 83 ismaintained. Leakage of specimen fluids from between the valve chamber101 and body 76 is prevented by O-rings 98 and 99, the intennal pressureof the system in actuality enhancing the sealing action of said O-rings.

Should any wear occur on either or both valve head 91 and constriction116 due to frictional engagement therebetween, the position of valvestem 88 may be relocated in respect of valve stem screw 83 by adjustingset screw 7 87. Since the valve stem, valve head, and valve seat areaccurately machined, any wear that takes place therebetween issubstantially uniform, and, therefore, a re-setting of set screw 83 issufiicient to maintain the apparatus in working order.

The complete system, shown schematically in FIG. 4,

may be enclosed in a suitable cabinet, or the like, not

In some embodiments it may be desirable to arrange for some of thecomponents to be compartmented into a separate enclosure, indicated bythe dotted lines 132, so that the processing of the specimens may takeplace in an environment which may be irradiated by ultraviolet orperfused with various materials, such as inert gases or bactericidal orgermicidal sprays in order to proted the specimens from contamination.Furthermore, enclosure 132 serves to protect the operator when certaintypes of infectious or otherwise dangerous materials are being processedby the apparatus.

Suitable leak-tight connections are provided in com- 'partment 132,,whereby the cooling medium may be introduced and withdrawn, theoperation of the pressure cell piston facilitated by a flexibleair-tight juncture 133 'andfor various external controls for setting thepressure value of one-way valve 14, and operating valve stem screw 83,and the like. Furthermore, such leak-tight connections serve not only toprevent contamination of the external controls and apparatus, but alsoserve to prevent the escape of contaminating materials into theatmosphere of the laboratory; It is claimed:

1. Cell fractionating apparatus comprising a valve assembly, first meansfor introducing a suspension of cells under pressure into said valveassembly, second means for said valve chamber and said expansionchamber, a mov- I able valve element in said valvevchambercooperatingwith said valve seat, and means in said body'for continuouslycirculating a cooling medium in said expansion chamber and in the regionof said valve seat.

3. Apparatus according to claim 2 wherein said valve seat comprises aconcave conoidal constriction in said valve chambenand said valveelement comprises a conical head that cooperates coaxiallywiththesurface of said constriction. V V

4. Apparatus according to claim 2 wherein the means for introducing thesuspension of cells into said valve chamber comprises an inlet channelin said body, an annular channel in said body communicating with saidinlet channel, and a circular array of radial ports in said bodyestablishing communication between said annular channel and the interiorof said valve chamber.

5. Apparatus according to claim 2 whereinsaid .valve element comprisesan elongated stem, a valve head on said stem cooperating with said valveseat, a valve stem screw threadably engaging said body, an axialbore insaid valve stem screw accommodating said valve stem-with a tight'runningfit, and a set screw in said valve stem screw -at the endof said boreand against which said valve stem is abutted, said set screw beingadjustable to establish the requisite position of said valve stem inrelation to said valve stem screw.

6. Cellfractionating apparatus according to claim 2 wherein said valveseat terminates in a small circular orifice at the juncture between saidvalve chamber and said expansion chamber, said expansion chamber beingconicalin shape and said orifice forming the smaller end of saidexpansion chamber.

7.Cell fractionating apparatus according toclaim 2, and furthercomprising an outlet bore in said body communicating with saidexpansionchamber, and outlet tube extending into said bore, the interior diameterof said bore being greater thanthe outside diameter of said tube, aninlet channel communicating with said bore for introducing a coolingmedium therethrough and into said expansion chambensaid tube serving asan:outlet for both the fractionated suspension of cells and saidcoolingmedium.

8. Cell fractionating apparatus comprising aspecimen supply source, acompression device connected to said supply source, a one-way valveconnected between said compression device and said source preventing thereturn of said specimen to said source, a valve apparatus to which saidcompression device delivers the specimen under pressure, an expansionchamber insaid valve apparatus in which the specimen undergoes rapiddecompression, means for cooling said compression device, and means forcontinuously circulating an inert cooling medium into said expansionchamber.

9. Cell fractionating apparatus comprising a' specimen supply source, acompressiondevice connected to said supply source, a one-way valveconnected between said compression device and said source preventing thereturn of said specimen to said source, a valve'apparatus to which saidcompression device delivers the specimen under pressure, an expansionchamberin said valve apparams in which the specimen undergoes rapiddecompression, an inert cooling medium, means for cooling said mediumand transmitting it to said compression device to cool the latter, meansfor thereafter re-cooling said me dium, and means forcontinuously-conducting said recooled medium into said expansion chamberfor cooling the latter.

10. Apparatus according to claim 9, and further comprising a receiver,and means for transmitting the decompressed specimen and said coolingmedium into said receiver. g

11. Cell fractionating apparatus comprising a valve body, a bottom platedetachably connected-to said body, a recess in said body,,a valve blockpositioned in said recess and held captive therein by said bottom plate,an integral tubular boss on said valve block, a second recess coaxialwith said first recess in said body accommodating said boss, a valvechamber in said boss and said block, a bore in said body coaxial withsaid valve-chamber, a valve, stem in said bore, a valve head on saidstem movable longitudinally insaid valve chamber, a valve seat at thelower end of said valve chamber, said valve head cooperating with saidvalve seat, an expansion chamber in said block communicating coaxiallywith said valve seat, and an exitbore in said bottom plate positionedcoaxially relative to said expansion chamber and communicatingtherewith, I I

12. Apparatus according to claim 11 and further comprising an annularrecess in said second recess-surrounding saidboss, an inlet passage insaid body communicating with said annular recess, and a plurality ofradially arrayedbores in said'boss establishing communication betweensaid annular recess and said valve chamber.

13. Apparatus according to claim 11', and further comprising a circularorifice between said valve seat and said expansion chamber, said orificebeing of substantially smaller diameter than said .valve chamber, saidexpansion chamber being conical in shape and said orifice forming thesmaller end thereof.

14. Apparatus according to claim 11, and further comprising an inletchannel in said bottom plate communicating with the bore in said bottomplate for introducing and circulating a cooling medium into saidexpansion chamber.

15. Apparatus according to claim 11, and further comprising an exit tubepositioned in the bore of said bottom plate, the inside diameter of saidbore being larger than the outside diameter of said exit tube, the innerend of said exit tube being positioned coaxially opposite the wider endof said expansion chamber, an inlet channel in said bottom platecommunicating with said bore for introducing and circulating a coolingmedium through said bore into said expansion chamber, said tube servingas exit means for materials expelled from said valve chamber and forsaid cooling medium.

16. The method of fractionating cells which comprises compressing asuspension of said cells, passing said compressed suspension through asmall orifice into an expansion chamber, cooling an inert gas,conducting said gas in a coil around the means for compressing saidsuspension of cells to absorb heat generated by the compression process,re-cooling said inert gas, and circulating said re-cooled gascontinuously into said expansion chamber to cool the latter and saidorifice.

17. Cell fractionating system comprising a body, a high pressurespecimen chamber in said body, an expansion chamber in said body axiallyaligned and communicating with said specimen chamber, an annular valveseat between said chambers, a valve movable axially within said specimenchamber and adapted to mate with said valve seat, said valve beingretractable from said seat to permit the specimen under high pressure insaid specimen chamber to escape into said expansion chamber, means forintroducing a cell specimen under high pressure into said specimenchamber, and means for continuously circulating a cold gas into andthrough said expansion chamber to cool the latter and to cool said valveseat.

18. Cell fractionating system comprising a body, a high pressurespecimen chamber in said body, an expansion chamber in said body axiallyaligned and communicating with said specimen chamber, an annular valveseat between said chambers, a valve movable axially within said specimenchamber and adapted to mate with said valve seat, said valve beingretractable from said seat to permit the specimen under high pressure insaid specimen chamber to escape into said expansion chamber, means forintroducing a cell specimen under high pressure into said specimenchamber, means for continuously circulating a coid gas into and throughsaid expansion chamber to cool the latter and to cool said valve seat,and an exit passage in said body axiallyv aligned and communicating withsaid expansion chamber, said cold gas and the fractionated specimenpassing together from said expansion chamber through said exit passage.I

19. Cell fractionating apparatus comprising a body, a valve chamber insaid body, an expansion chamber in said body axially aligned andcommunicating with said valve chamber, a constriction between said valvechamber and said expansion chamber, a valve movable axially within saidvalve chamber, a valve head on said valve, a portion of saidconstriction forming a valve seat for said valve head to provide anannular seal between said valve chamber and said expansion chamber, aportion of said valve head extending through said constriction, meansfor introducing cells in suspension under high pressure into said valvechamber, and means for continuously circulating a cold gas into saidexpansion chamber to cool the latter and to cool said constriction, saidvalve seat and the portion of the valve head extending through saidconstriction.

References Cited by the Examiner UNITED STATES PATENTS 2,318,693 5/43Joyce et al. 2411 2,873,220 2/59 Brownell et a1. 241-2 2,928,614 3/60Emanuel et al. 2411 1. SPENCER OVERHOLSER, Primary Examiner.

JOHN C. CHRISTIE, Examiner.

1. CELL FRACTIONATING APPARATUS COMPRISING A VALVE ASSEMBLY, FIRST MEANSFOR INTRODUCING A SUSPENSION OF CELLS UNDER PRESSURE INTO A SAID VALVEASSEMBLY, SECOND MEANS FOR EMITTING FRACTIONATED CELLS FROM SAIDASSEMBLY, A VALVE POSITIONED BETWEEN SAID FIRST MEANS AND SAID SECONDMEANS, AN EXPANSION CHAMBER BETWEEN SAID VALVE AND SAID SECOND MEANS,AND THIRD MEANS CONNECTED TO SAID VALVE ASSEMBLY FOR CONTINUOUSLYCIRCULATING A COOLING MEDIUM IN SAID EXPANSION CHAMBER AND IN SAIDVALVE.